Mammalian cells remove misfolded proteins by degradation mediated by the ubiquitin (Ub) proteasome pathway and autophagy. The first step in the development of neuronal dysfunction or neuronal death is the lack of a response to misfolded proteins. Dysfunction in the Ub proteasome pathway in neurons leads to the accumulation of aggregates and plaques in neurons. Ubiquitylation of proteins not only controls the half-lives of proteins but also acts as a regulatory modification, e.g., histone ubiquitylation. Due to the dynamic nature of Ub conjugation and deconjugation, ubiquitylated proteins are extremely difficult to isolate and study. Identifying a protein with one particular Ub post translational modification out of the pool of modified proteins becomes nearly impossible without selective tools. A further complication is the presence of multiple types of Ub-Ub linkages in polyubiquitin chains. Ubiquitin is attached, via isopeptide bonds, to lysine residues in the target protein. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs, again through the formation of isopeptide bonds between the C-terminus of one Ub and any of seven (7) lysines, or N-terminus of the target Ub. Ub chains with different linkages convey different messages to cells and, hence, determine the ultimate fate of the protein -- degradation, translocation, and oxidative stress response, to name a few. The precise information encoded in the various chain linkages is largely unknown due to a lack of reagents that selectively recognize these linkages. The goal of this proposal is to develop tools that allow the selective identification, quantification, and isolation of proteins modified by mono and polyubiquitin chains containing difficult to study linkages. This will be accomplished using a novel Ub binding microarray that contains all of the known Ub binding motifs from various proteins. In Phase I, we will identify and characterize novel Ub binding domains (UbDs) exhibiting selectivity for monoubiquitin, monoubiquitylated substrates and specific polyubiquitin linkages. Given the fact that dysregulation of the Ub proteasome pathway and autophagy have been strongly implicated as a first step in neurodegenerative diseases, the development of sensitive and novel chain selective tools will lead to early diagnosis of Alzheimer?s and Parkinson?s diseases (phase II).